Antigens are processed by antigen-presenting cells. In order to trigger an efficient immune response, antigens must first be processed by an antigen-presenting cell (APC) that degrades the antigen and “presents” the resulting antigen fragments to other cells involved in the immune response. Macrophages are among the most commonly encountered type of APC; other examples include dendritic cells in the spleen and Kupffer cells in the liver. Macrophages readily engulf foreign particles and cells by the process of phagocytosis. Although all macrophages are avid phagocytes, only some can process foreign antigens in a way that stimulates an immune response. The macrophages that perform this function carry specialized plasma membrane glycoproteins called major histocompatibility complex (MHC) molecules. In man the MHC is known as HLA. MHC molecules reside only on the surfaces of cells involved in immune responses.
The processing of foreign antigens by macrophages and other APCs involves four main stages. First, antigens are taken into the cell by a relatively nonspecific form of endocytosis that brings all molecules dissolved in the extracellular fluid into the cell. Next, the internalized endocytic vesicles fuse with lysosomes, whose hydrolytic enzymes degrade the foreign antigens into small fragments. In the case of ingested protein antigens, the process yields peptide fragments about 10–20 amino acids long. Third, the resulting fragments become bound to MHC class II molecules, which possess an antigen-binding cleft that is specifically designed to bind antigen fragments. Finally, the foreign antigen-MHC II complex is transported to the cell surface for “presentation” to other cells of the immune system. The importance of the preceding pathway has been demonstrated by treating cells with inhibitors that block endocytosis or the lysosomal degradation of antigens; under such conditions, an immune response is inhibited.
The pathway that was just described is used in the processing of exogenous antigens (i.e. antigens that have been taken up from outside the cell. Immune responses can also be triggered by endogenous antigens that originate within the cells of the host organism. For example, cells that have become infected by a virus usually manufacture foreign proteins encoded by viral genes. Fragments derived from the viral proteins become bound to a different type of MHC molecule called an MHC class I molecule. Peptide antigens destined to bind to MHC class I molecules are produced by protein cleavage in the cytosol and are then transported into the lumen of the endothelial reticulum, where the fragments bind to class I molecules associated with the endothelial reticulum membrane. The resulting antigen-MHC I complex is then transported to the cell surface, where its presence can trigger an immune response against the infected cell. (Peptide fragments derived from a cell's own normal proteins can also bind to MHC class I molecules and be presented at the cell surface, but such “self” antigen-MHC complexes do not usually elicit an immune response.)
Activation of naive T-cells requires recognition of a foreign antigenic fragment bound to a self MHC molecule but this is not on its own sufficient for activation. It also requires the simultaneous delivery of a co-stimulatory signal by a specialized antigen-presenting cell. It has been believed that only professional antigen-presenting cells (APCs) are able to express both classes of MHC molecules as well as the co-stimulatory surface molecules that drive the clonal expansion of naive T-cells and their differentiation into armed effector T-cells. The activation of naive T-cells on initial encounter with antigen on the surface of a professional antigen-presenting cell is often called priming, to distinguish it from the responses of armed effector T-cells to antigen on their target cells, and the responses of primed memory T-cells.
Effector T-cells are triggered when their antigen-specific receptors (T cell receptors) and either the CD4 or the CD8 co-receptors bind to peptide-MHC complexes. But ligation of the T-cell receptor and co-receptor does not, on its own, stimulate naive T-cells to proliferate and differentiate into armed effector T-cells. The antigen-specific clonal expansion of naive T-cells requires a second, co-stimulatory, signal which is delivered by the same antigen-presenting cell on which the T-cell recognizes its specific antigen.
The best characterized co-stimulatory molecules on antigen-presenting cells are the structurally related glycoproteins B7-1 and B7-2. These are homodimeric members of the immunoglobulin superfamily found exclusively on the surface of cells capable of stimulating T-cell growth. The receptor for B7 molecules on the T-cell is CD28, yet another member of the immunoglobulin superfamily. Ligation of CD28 by B7-1 or B7-2 or by anti-CD28 antibodies will co-stimulate the growth of naive T-cells, while antibodies to the B7 molecules, which inhibit their binding to CD28, inhibit T-cell responses.
On naive T-cells, CD28 is the only receptor for B7-1 and B7-2. Once T-cells are activated, however, they express an additional receptor called CTLA-4, which binds B7 molecules with a higher affinity than does CD28. CTLA-4 closely resembles CD28 in sequence, and the two molecules are encoded by closely linked genes. CTLA-4 binds more avidly than CD28 but appears to play a negative role in the activation of the T-cell expressing it. The activated progeny of a naive T-cell become less sensitive to stimulation by antigen than the naive T-cells. This may help to limit the early proliferative response of the T-cells to antigen and B7 molecules on the surface of antigen-presenting cells. Although other molecules have been reported to co-stimulate naive T-cells, to date only B7-1 and B7-2 binding to CD28 has been shown definitively to provide co-stimulatory signals in normal immune responses.
The requirement for simultaneous delivery of antigen-specific and co-stimulatory signals in the activation of naive T-cells means that only professional antigen-presenting cells can initiate T-cell responses. This is important because not all potentially self-reactive T-cells are deleted in the thymus: peptides derived from proteins made only in specialized cells in the peripheral tissues may not be encountered during the negative selection of thymocytes. Self tolerance could be broken if naive, autoreactive T-cells could recognize self antigens on tissue cells and then be co-stimulated by a professional antigen-presenting cell, either locally or at a distant site. Thus, the requirement that the same cell presents both the specific antigen and the co-stimulatory signal plays an important part in preventing destructive immune responses to self tissues. Indeed, antigen binding to the T-cell receptor in the absence of co-stimulation not only fails to activate the cell but also leads to a state called anergy, in which the T-cell becomes refractory to activation.
For many years, immunologists studying how the immune system recognizes foreign molecules have focused on the molecular mechanisms by which T-cells recognize peptide antigens. Zeng et al., Science. 277:339 (1997) present the three-dimensional structure of murine CD1d, a representative of a family of conserved mammalian proteins that are somewhat related to MHC molecules. This structure reinforces the view that CD1 proteins bind and present antigen in a way that allows T-cells to be activated by lipid and glycolipid antigens.
The CD1 genes are located on a different chromosome than the MHC in humans. These are nonpolymorphic proteins with only about 30% homology to MHC class I or II molecules. Despite this marked divergence from MHC structure, a role for CD1 in antigen presentation was shown by the finding that CD1 expression on antigen-presenting cells was required for the responses of certain T-cell clones to Mycobacterium tuberculosis. Further studies in this system led to the finding that the mycobacterial antigens recognized by CD1 restricted T-cells are not peptides, but instead are lipids (mycolic acids) and glycolipids found in the cell walls of these bacteria.
Mesenchymal stem cells are the formative pluripotential blast cells found inter alia in bone marrow, blood, dermis and periosteum that are capable of differentiating into any of the specific types of mesenchymal or connective tissues (i.e. the tissues of the body that support the specialized elements; particularly adipose, osseous, cartilaginous, elastic, and fibrous connective tissues) depending upon various influences from bioactive factors, such as cytokines. Although these cells are normally present at very low frequencies in bone marrow, a process has been discovered for isolating, purifying, and greatly replicating these cells in culture, i.e. in vitro. See, Caplan and Haynesworth, U.S. Pat. No. 5,486,359.
In order to isolate human mesenchymal stem cells, it is necessary to isolate rare pluripotent mesenchymal stem cells from other cells in the bone marrow or other MSC source. Bone marrow cells may be obtained from iliac crest, femora, tibiae, spine, rib or other medullary spaces. Other sources of human mesenchymal stem cells include embryonic yolk sac, placenta, umbilical cord, fetal and adolescent skin, and blood.
Isolated human mesenchymal stem cell compositions serve as the progenitors for multiple mesenchymal cell lineages including bone, cartilage, ligament, tendon, adipose, muscle, stroma, dermis and other connective tissues. These isolated mesenchymal cell populations have the ability to expand in culture without differentiating, and have the ability to differentiate into specific mesenchymal lineages when either induced in vitro or placed in vivo at the site of damaged tissue. To date, they have conventionally been associated with their usefulness in connective tissue repair.